EP1693469B1

EP1693469B1 - Detecting DNA sequence variations

Info

Publication number: EP1693469B1
Application number: EP06076004A
Authority: EP
Inventors: Stephen Charles Oxford Gene Technology Case-Green; Clare Elizabeth Oxford Gene Technology Pritchard; Edwin Mellor Oxford Gene Technology Southern
Original assignee: Oxford Gene Technology IP Ltd
Current assignee: Oxford Gene Technology IP Ltd
Priority date: 1995-04-07
Filing date: 1996-04-09
Publication date: 2010-08-25
Anticipated expiration: 2016-04-09
Also published as: EP1693469A3; US6770751B2; US6150095A; GB9507238D0; JP3908784B2; US20050266408A1; DE69638248D1; US6307039B1; JP2006246897A; US7192707B2; JPH11503019A; EP1308523A2; US20020106665A1; DE69636843T2; US8084211B2; WO1996031622A1; DE69633974T2; EP1308523B1; DE69636843D1; DE69633974D1

Description

Detection of variation in DNA sequences forms the basis of many applications in modern genetic analysis: it is used in linkage analysis to track disease genes in human pedigrees or economically important traits in animal and plant breeding programmes; it forms the basis of fingerprinting methods used in forensic and paternity testing [Krawczak and Schmidtke, 1994]; it is used to discover mutations in biologically and clinically important genes [Cooper and Krawczak, 1989]. The importance of DNA polymorphism is underlined by the large number of methods that have been developed to detect and measure it [Cotton, 1993]. Most of these methods depend on one of two analytical procedures, gel electrophoresis or molecular reassociation, to detect sequence variation. Each of these powerful procedures has its drawbacks. Gel electrophoresis has very high resolving power, and is especially useful for the detection of variation In the mini- and microsatellite markers that are used in linkage analysis and fingerprinting: it is also the method used to analyse the variation found in the triplet repeats that cause a number of mutations now known to be the cause of around ten genetic disorders in humans [Willems, 1994]. Despite its great success and widespread use, gel electrophoresis has proved difficult to automate: even the systems which automate data collection require manual gel preparation; and as samples are loaded by hand, it is easy to confuse samples. The continuous reading electrophoresis machines are expensive, and manual analysis is technically demanding, so that its use is confined to specialised laboratories which have a high throughput. Furthermore, difficulties in measuring fragment size preclude rigorous statistical analysis of the results.
By contrast, oligonucleotide hybridisation lends itself to automation and to quantitative analysis [Southern et al., 1992], but it is not well suited to the analysis of variation in the number of repeats in the micro- and minisatellites, as the small fractional change in the number of repeats produces a barely detectable change in signal strength; and of course it would not be possible to distinguish two alleles in the same sample as each would contribute to a single intensity measurement. Thus, many different combinations of alleles would produce the same signal. Present hybridisation methods are much better suited to analysing variation in the DNA due to point mutation - base substitution deletions and insertions, for which it is possible to design allele specific oligonucleotides (ASOs) that recognise both the wild type and the mutant sequences [Conner et al., 1983]. Thus it is possible in principle, In a relatively simple test, to detect all possible genotypes. However, a problem that arises in practice in the use of oligonucleotide hybridisation is that in some cases the extent of reassociation is barely affected by a mismatched base pair.

THE INVENTION

The invention describes a general approach which can be applied to all forms of variation commonly used as DNA markers for genetic analysis. It combines sequence-specific hybridisation to oligonucleotides, which are tethered to an array of individual beads, with enzymatic reactions which enhance the discrimination between matching and non-matching duplexes, and at the same time provide a way of attaching a label to indicate when or which reaction has taken place, chain extension by DNA dependent DNA polymerases is dependent on perfect matching of sequences at or around the point of extension or joining. As we shall show, there are several ways in which these enzymes can be used with sequence-specific oligonucleotides to detect variation in target sequences.
In all cases, the sequence to be analysed, the target sequence, will be available as a nucleic acid molecule, and may be a DNA molecule produced, for example, by the polymerase chain reaction. However, the methods are not confined to analysis of DNA produced in this way. The target sequence is first captured by hybridisation to oligonucleotides which are tethered to individual beads; for example, the oligonucleotides may be synthesised in situ as described [Maskos and Southern, 1992]; or they may be presynthesised and then coupled to the surface [Khrapko et al. 1991]_.
In one aspect of the invention the novelty arises from the exploitation of enzymes in combination with substrates or primers tethered to individual heads.
In one aspect the invention provides a method of analysis which comprises: providing a polynucleotide target including a nucleotide at a specified position, and an array of oligonucleotide probes, tethered to a individual beads, said probe being complementary to the target and terminating are base before the said specified position; and performing the steps:

a) incubating the target with the probe to form a duplex,
b) incubating the duplex under chain extension conditions with at least one labelled nucleotide,
c) and monitoring chain extension in b) as an indication of the nucleotide at the specified position in the target.

A polynucleotide target is provides, in solution when the probe is tethered to a support, and may be DNA or RNA. This polynucleotide target is caused to hybridise with an oligonucleotide probe. The term oligonucleotide is used here, as common terminology for the primers and substrates commonly utilised by polymerase and ligase enzymes. However, the term is used in a broad sense to cover any substance that serves as a substrate for the enzymes, including single stranded chains of short or moderate length composed of the residues of nucleotides or of nucleotide analogues, and also longer chains that would ordinarily be referred to as polynucleotides.
The probes may be tethered to individual beads preferably by a covalent linkage and preferably through a 5' or 3'terminal nucleotide residue. An array of oligonucleotide probes are tethered at spaced locations, on individual beads.
In another aspect there is described an array of oligonucleotides, for analysing a polynucleotide target containing a variable sequence, in which each component oligonucleotide i) comprises a sequence complementary to the target including an expected variant of the target, and ii) is tethered to an individual bead in a chemical orientation which a) permits duplex formation with the target, and b) permits chain extension only when the sequence of the oligonucleotide matches the variable sequence of the target.
In another aspect there is described an array of oligonucleotides in which different oligonucleotides occupy different individual beads and each oligonucleotide has a 3' nucleotide residue through which it is covalently tethered to a support and a 5' nucleotide residue which is phosphorylated.
Reference is directed to the accompanying drawings in which each of Figures 1 to 6 is a series of diagrams illustrating a method according to the invention.

Figure 1 shows detection of point mutation by single base extension.
Figure 2 shows detection of point mutation by hybridisation to allele specific oligonucleotides and chain extension.

DETAILED DESCRIPTION

Detection of point mutation

I. Single base-specific extension of tethered primers.

In this application, the tethered oligonucleotide terminates at a position one base before the variable base in the target sequence (Fig.1). A nucleotide precursor triphosphate or dideoxyribonucleotide triphosphate, labelled, for example with a fluorescent tag, is added in the presence of a nucleic acid synthesising enzyme which requires a specific template in order to incorporate the complementary base. In the case of DNA polymerase, the labelled base will be incorporated from a deoxyribonucleotide precursor only if the precursor base is complementary to the base in the target sequence. Thus, mutants will give a negative result.

II. Chain extension from tethered ASOs.

In this case, the tethered oligonucleotide terminates in a base which is complementary to the variable base in the target sequence. Labelled precursor nucleoside triphosphates and polymerase are added. Polymerisation takes place only if the last base of the primer is complementary to the variable base in the target (Fig.2). Thus, mutants will give a negative result.

EXPERIMENTAL SUPPORT FOR THE CLAIMS

Properties of DNA Polymerases

Most DNA polymerases, reverse transcriptases, some DNA dependent RNA polymerases can use as substrates one or more oligonucleotides which are bound to a long DNA strand through Watson-Crick base pairing. In the case of polymerases, an oligonucleotide is used as a primer to which the first base in the growing chain is added. It is this property that makes the enzyme useful for the detection of DNA sequence variation; in particular, the requirement for specific base pairing at the site of extension complements the sequence discrimination that is already provided by the Watson-Crick pairing between the oligonucleotide and the target sequence that is needed to form a stable duplex. Thus, it has been found that discrimination by hybridisation alone is most sensitive if the variant base(s) is (are) close to the middle of the oligonucleotide. By contrast, for the enzymes, discrimination is highest if the variant mismatching bases are close to the end where the extension or join takes place. Together, hybridisation under stringent conditions and enzymatic extension provide greater discrimination than either alone, and several methods have been developed to exploit this combination in systems for genetic analysis [references in cotton, 1993].
The invention described here employs an array of oligonucleotides coupled individual beads, so that the advantages of working in mixed phase are brought to all steps: hybridisation, enzymatic extension or joining, and detection. This provides great sensitivity and convenience. This enables many different sequences to be analysed together, in a single reaction; this also ensures that all reactions are carried out under identical conditions, making comparisons more reliable.

Support-bound Oligonucleotides

Two different methods have been developed for making oligonucleotides bound to individual beads; they can be synthesised in situ, or presynthesised and attached to the bead. In either case, it is possible to use the support-bound oligonucleotides in a hybridisation reaction with orgonucleotides in the liquid phase to form duplexes; the excess of oligonucleotide in solution can then be washed away. Hybridisation can be carried out under stringent conditions, so that only well-matched duplexes are stable. When enzymes are to be used, the chemical orientation of the oligonucleotide is important: polymerases add bases to the 3' end of the chain. Oligonucleotides tethered to the beads through either end can be made in situ by using the appropriate phospharamidite precursors [references in Beaucage and lyer, 1992]; or presynthesised oligonucleotides can be fixed through appropriate groups at either end. We will demonstrate that oligonucleotides can be phosphorylated at the 5' end in situ using ATP and polynucleotide kinase, or they may be phosphorylated chemically [Horn and Urdea, 1986].

Tethered Oligonucleotides as Substrates for DNA Modifying Enzymes

The applications envisaged here require that the oligonucleotides tethered to the solid substrate can take part in reactions catalysed by DNA polymerases.

DNA polymerase

The M13 sequencing primer-5'-GTAAAACGACGGCCAGT-3' - attached to aminated polypropylene through its 5' end was synthesised as described. A solution of M13 DNA (single-strand, replicative form, 0.1 µl, 200 ng/µl) was applied in two small spots to the surface of the derivatised polypropylene. A solution containing three non-radioactive deoxyribonucleotide triphosphates, dATP, dGTP, TTP (10 µmol each), α³²P-dCTP (10 µCl), Taq DNA polymerase and appropriate salts, was applied over a large area of the polypropylene, including the area where the M13 DNA had been spotted. The polypropylene was incubated at 37°C for 1 hr in a vapour saturated chamber. It was then washed in 1% SDS at 100°C for one minute, and exposed to a storage phosphor screen for one minute and scanned in a phosphorimager. The regions where the DNA had been applied showed a high level of radioactivity, against a low background where no DNA had been applied. This experiment shows that oligonucleotides tethered to a solid support can act as primers for DNA-dependent synthesis by DNA polymerase, as required for applications using this enzyme for mutation detection.
Experiments described below show that both polynucleotide kinase and DNA ligase can be used to modify oligonucleotides tethered to a solid support. There are several ways in which phosphorylated oligonucleotides and the ligase reaction can be used to detect sequence variation.

Methods for Making Arrays of Sequence Variants.

1. Allele specific oligonucleotides for point mutations.

It will be necessary to use an array of oligonucleotides tethered to a individual beads for example, glass spheres, or magnetic beads. In this case the reactions could be carried out in tubes, or in the wells of a microtitre plate. Methods for both synthesising oligonucleotides and for attaching presynthesised oligonucleotides to these materials are known [Stahl et al., 1988]. Methods for making arrays of ASO's representing point mutations were described In patent application WO89/0977 and in Maskos and Southern ( 1993 ). We also demonstrated how oligonucleotides tethered to a individual beads could distinguish mutant from wild type alleles by molecular hybridisation.
For the present invention, the same methods could be used to create oligonucleotide arrays of ASOs, but in order that they can be used as substrates for the enzymes, they need to be modified; for extension by polymerase it will be necessary to attach to oligonucleotides to the solid substrate by their 5' ends.

2. Arrays for scanning regions for mutations.

It is often desirable to scan a relatively short region of a gene or genome for point mutations: for example, many different sites are mutated in the CFTR gene to give rise to cystic fibrosis: similarly, the p53 tumour suppressor gene can be mutated at many sites. The large numbers of oligonucleotides needed to examine all potential sites in the sequence can be made by efficient combinatorial methods [Southern et al., 1994]. A modification of the protocol could allow such arrays to be used in conjunction with enzymes to look for mutations at all sites in the target sequence.

EXAMPLE 1

Analysis by ligation and polymerisation

A strip of the array (30mm x 2mm) from Example 2 was added to a solution of 200 pmols of the target oligonucleotide 5' cacagactccatgg(tgaa)₆tgagggaaataag, 200 pmol of oligo 5' ccatggagtctgtg (chemically phosphorylated at the 5' end) with buffer and salts according to the suppliers instructions, the total volume being 243 µl. The solution was heated to 65°C and cooled to 37°C over a period of 30 mins. 7 µl of Tth DNA ligase was added and the reaction mixture heated at 34°C for 17hrs. The strip was removed and added to a solution of 8 mM DTT, 3.3 pmol ³²P alpha dTTP, 13 units sequenase version 2.0 and buffer and salts according to the suppliers instructions. The total volume was 250 µl. After heating at 37°C for 3 hrs the strip was removed from the reaction solution, washed in TE buffer, blotted dry and exposed to a storage phosphor screen from which an image of the radioactivity was taken. The results showed counts equal to background over the area of the array where repeats were equal in length or greater than the repeat length of the target, with 20 times the signal in the areas where the repeat length of the array was shorter than the repeat length in the target.

EXAMPLE 2

Analysis by polymerisation

Two types of polymerase analysis were carried out where reporter nucleotides were chosen, in one case to identify the correct repeat length, and in the other case to identify shorter repeat lengths. This is made possible when the repeat sequence comprises less than all four bases.
In the former case a base is chosen which is present in the repeat sequence and is different from the first base in the flanking sequence. In the latter case a base is chosen to be complementary to the first base in the flanking sequence which is absent from the repeat.
A strip of the array from Example 2 (30mm x 2mm) was added to a solution of 500 pmols of the target oligonucleotide 5' cacagactccatgg(tgaa)₆tgagggaaataag in buffer and salts at a concentration 1.09 times the suppliers instructions, the total volume being 275 µl. The solution was heated to 75°C for 5 minutes and cooled to 37°C over a period of 25mins. The solution was removed and added to 3.3 pmols ³²P alpha dCTP, 5 µl 1M DTT. 13 units of Sequenase version 2.0 and water to give a final volume of 295 µl. This solution was added to the array and heated at 37°C for 15hrs 40mins. The polypropylene strip was removed, washed in water and exposed to a storage phosphor screen. The results showed counts of 5 times more for the correct sequence than shorter sequences and twice for the correct sequence compared with longer repeats.
A similar strip of the array (30mm x 2mm) was added to a solution of 500 pmols of the target oligonucleotide 5' cacagactccatgg(tgaa)₆tgagggaaataag in buffer and salts 1.09 times the suppliers instructions, the total volume being 275 µl. The solution was heated to 75°C for 5 mins and cooled to 37°C over a period of 25 mins. The solution was removed and added to 3.3 pmols ³²P alpha dTTP, 5 µl 1M DTT, 13 units of Sequenase version 2.0 and water to give a final volume of 295 µl. This solution was added to the array and heated at 37°C for 15hrs 40mins. The polypropylene strip was removed, washed In water and exposed to a storage phosphor screen. The results showed counts of 4.5 times more for the shorter array sequences than the correct and longer repeat lengths.

REFERENCES

1. Beaucage, S. L. and lyer, R. P. (1992). Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48: 2223-2311.
2. Conner, B. J., Reyes, A. A., Morin, C., Itakura, K., Teplitz, R. L., and Wallace, R. B. (1983). Detection of sickle cell β' globin allele by hybridization with synthetic oligonucleotides. Proc. Natl. Acad. Sci. USA 80: 278-282.
3. Cooper, D. N., and Krawczak, M. (1989). The mutational spectrum of single base-pair substitutions causing human genetic disease: patterns and predictions. Hum. Genet. 85: 55-74.
4. Cotton, RG, (1993) Current methods of mutation detection. Mutation Research 285: 125-144.
5. Horn, T., and Urdea, M. (1986) Chemical phosphorylation of oligonucleotides. Tetrahedron Letters 27: 4705.
6. Khrapko, K. R., Lysov, Yu. P., Khorlyn, A. A., Shick, V. V., Florentiev, V. L., and Mirzabekov. (1989). An oligonucleotide hybridization approach to DNA sequencing. FEBS Lett. 256: 118-122.
7. Krawczak M. and Schmidtke, J. (1994). DNA fingerprinting. BIOS Scientific Publishers.
8. Maskos, U., and Southern, E.M., (1993) A novel method for the analysis of multiple sequence variants by hybridisation to oligonucleotides. Nucleic Acids Research, 21: 2267-2268.
9. Pillai, V. N. R.(1980). Photoremovable protecting groups in organic chemistry Synthesis 39: 1-26.
10. Southern, E. M. (1988). Analyzing Polynucleotide Sequences. International Patent Application PCT/GB89/00460 .
11. Southern, E.M., Maskos, U. and Elder, J.K. (1992). Analysis of Nucleic Acid Sequences by Hybridization to Arrays of Oligonucleotides: Evaluation using Experimental Models. Genomics 12: 1008-1017.
12. Southern. E. M., Case-Green, Elder, J.K. Johnson, M., Mir, K.U., Wang, L., and Williams, J.C. (1994). Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids. Nucleic Acids Res. 22:, 1368-1373.
13. Stahl, S., Hultman, T., Olsson, A., Moks, T.D and, Uhlen, M. (1988) Solid phase DNA sequencing using the biotin-avidin system. Nucleic Acids Res. 16: 3025-38.
14. Veerle, A.M.C.S., Moerkerk, P.T.M.M., Murtagh, J.J., Jr., Thunnissen, F.B.J.M (1994) A rapid reliable method for detection of known point mutations: Point-EXACCT. Nucleic Acids Research 22: 4840-4841.
15. Vimekas, B., Liring, G., Pluckthon, K., Schneider, C., Wellhofer, G. and Moroney, S. E. (1994) Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Research 22: 5600-5607.
16. Willems, P.J. (1994) Dynamic mutations hit double figures. Nature Genetics 8: 213-216.

DNA sequences of triplet repeats

Condition	Repeat	Normal	Preexpansion	Expanded	Reference
FRAXA	CGG/CCG	10-50	38-50	200-1000	Verkerk et al. 1991
FRAXE	CGG/CCG			200-1000	Knight et al. 1993
FRAXF	GCC/CGG	6-18	?	300-500	Parrish et al. 1994
FRAX16A	CGG/CCG			1000-2000	Nancarrow et al. 1994
SBMA	CAG	11-31	?	40-62	Tilley et al. 1994
Huntington	CAG	11-34	30-34	42-100	Huntington group 1993
SCA1	CAG	25-36	?	43-81	Orr et al. 1993
DRPLA/HRS	CAG			≤100	Burke et al. 1994
Machado-Joseph	CAG/CTG	∼26	?	68-79	Kawaguchi et al. 1994

SEQUENCE LISTING

<110> OXFORD GENE TECHNOLOGY IP LIMITED
<120> DETECTING DNA SEQUENCE VARIATIONS
<130> P043743EP
<150> GB-9507238.5
<151> 1995-04-07
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Claims

A method of analysis which comprises; providing a polynucleotide target including a nucleotide at a specified position, and an array of oligonucleotide probes, tethered to individual beads, wherein a probe in said array is complementary to the target and terminates one base before the said specified position; and performing the steps:
a) incubating the target with the probes to form a duplex,

b) incubating the duplex under chain extension conditions with at least one labelled nucleotide ; and

c) monitoring chain extension in b) as an indication of the nucleotide at the specified position in the target.
The method of claim 1, wherein different labelled nucleotides used in step (b) are labelled differently.
The method of claim 1, wherein the labelled nucleotide used in step (b) is a deoxyribonucleotide triphosphate, a dideoxyribonucleotide triphosphate or a nucleotide precursor triphosphate.
The method of any one of claims 1 - 3, wherein a nucleotide used in step (b) is labelled with fluorescent tag.
The method of any one of claims 1 - 4, wherein step (b) involves single base-specific extension.
The method of any one of claims 1 - 4, wherein the oligonucleotide probes are attached to the beads by their 5'ends.
The method of any one of claims 1 - 4, wherein the probes are tethered by a covalent linkage.
The method of any one of claims 1 - 4, wherein an enzyme is used in step (b).
The method of claim 8, wherein an enzyme is a polymerase.
The method of claim 8, wherein the enzyme is a DNA polymerase, a reverse transcriptase, or a RNA polymerase.
The method of claim 8, wherein the enzyme is a Taq polymerase or a Thermosequenase.
The method of any one of claims 1-4, wherein the probes are synthesised in situ.
The method of any one of claims 1-4, wherein the probes are presynthesised.